1. Field of the Invention
The focus of the present invention is an effective anti-poxvirus drug for use in treating or preventing human disease caused by pathogenic poxviruses. More particularly, the present invention relates to antiviral drugs that target the poxvirus proteinase responsible for core protein maturation, a step which is absolutely essential for the production and spread of infectious virions.
2. Description of the Related Art
The specter of bioterrorism has cast a pall over modern society. The ability to grow and manipulate microorganisms has provided biomedical scientists with a marvelous set of new abilities to develop therapies and drugs to combat both infectious and genetic diseases. Unfortunately these same technologies have a dark side. With very little sophisticated equipment required, the tools of microbiology and genetic engineering allow individuals or organizations to readily prepare large quantities of pathogenic microorganisms for deliberate dispersal into the environment. Unlike conventional weapons, pathogenic microorganisms have the ability to replicate and spread, they do not distinguish between the soldier and the civilian, and once released, they can not be recalled or controlled.
Whereas biological warfare concerns were originally limited to their potential deployment during conflicts between the armed forces of industrialized nations, the escalating threat of terrorism and domestic violence now raises the possibility that these agents may be used for this purpose as well. Amongst the biological agents that are believed to pose the greatest threat in this regard are Bacillus anthracis (anthrax), Yersinia pestis (bubonic plague), Francisella tularensis (tularemia), Coxiella burnetti (Q fever), hemorrhagic RNA viruses (e.g., Dengue, Marburg, Ebola, and Venezuelan Equine Encephalitis), and smallpox. While deliberate introduction of any of these agents would be devastating, the one which holds the greatest potential for harming the general population is smallpox, or a related genetically-engineered orthopoxvirus.
Smallpox was the most destructive disease in recorded history. It is estimated to have killed, crippled or disfigured nearly 1/10 of all humankind. In the 20th century alone, more than 300 million people succumbed to the disease. Variola virus, the causative agent of smallpox, is extremely infectious, causing obvious disease in most susceptible individuals that it comes into contact with.
Smallpox is usually contracted by inhalation of aerosol droplets spread by infected individuals. Following a 10–14 day prodrome period, frank disease erupts which is characterized by high fever and malaise. Two to three days later the patient's temperature drops and a systemic rash appears, characterized by the prototypic pustular lesions which can last 2–3 weeks. With the more virulent forms of smallpox, such as that caused by Variola major, as many as 30% of infected individuals die. Those that recover are scarred for life.
The disease has a number of features that make it “ideal” as an agent of bioterrorism: 1) the virus is easy to grow, can be lyophilized and does not require a cold chain to maintain viability; 2) the infection is spread by the respiratory route; 3) the prolonged prodrome period, combined with the ease of modern travel, allows the infected individual to spread the disease widely; 4) the resultant scarring has lasting psychological consequences; and 5) the virus is very stable in the environment after it has been shed, making decontamination difficult.
Although smallpox is of paramount concern, other potential orthopoxvirus pathogens should not be neglected. For example, there have been several recent incursions of monkeypox virus into the human population, although the human-human spread seems limited thus far. Another, more chilling, scenario is the possibility that a laboratory strain of vaccinia virus or cowpox virus could be genetically engineered to produce a toxin to convert it into a potent pathogen. Fortunately, the orthopoxviruses are highly related at the DNA level (e.g. 90% between variola and vaccinia) making it likely that any antiviral agent developed would inhibit the replication of this entire group of viruses.
Smallpox is no longer present in the natural environment, with the few known remaining laboratory stocks of variola virus slated for destruction in the near future. The eradication of smallpox was possible because it is a human-specific disease with no animal reservoirs, there was a single serotype, and the attenuated vaccine developed by Jenner effectively stimulates both the cellular and humoral arms of the immune system, providing long-lasting immunity. Smallpox was eliminated from the U.S. in the 1960's, and routine prophylactic immunization was discontinued in 1973.
The subsequent 30 years have produced a population that is immunologically naive and highly susceptible to orthopoxvirus infection. Due to the small but significant risk of serious complications from vaccination, especially in those immunocompromised due to infection by HIV or other agents, mass immunization of the populace is not advisable. Nevertheless, in the event that an infectious orthopoxvirus were introduced into the population, the live vaccine would be an effective weapon in limiting the spread of the disease once it had been diagnosed.
Vaccination before exposure, or within 2–3 days after exposure, affords almost complete protection. Vaccination as late as 4–5 days post-exposure can protect against death. Unfortunately, in the event of a deliberate introduction of the infectious agent into a heavily populated area, there would be a large reservoir of infected individuals by the time the sentinel cases were diagnosed. Furthermore, relatively limited stocks of the vaccine are available. For these reasons, as a protective measure, it is imperative that an effective anti-orthopoxvirus drug be available to treat those individuals exposed to the virus who have insufficient time to produce protective immunity and to help stem an epidemic.
The only well-known drug which is effective at inhibiting orthopoxvirus infections in cultured cells is methisazone or IBT (N-methylisatin β-thiosemicarbazone). However, this drug is of limited value in treating infected humans. Cidofovir has been demonstrated to be a potent anti-orthopoxvirus inhibitor, but it has severe side effects and must be delivered by injection. Thus, no safe, effective, orally-administrable anti-orthopoxvirus drug is currently available.
General Overview of Viral Proteolysis.
The term “limited proteolysis” was first introduced by Linderstrom-Lang and Ottesen to describe reactions in which the peptide bonds in a polypeptide are selectively hydrolyzed, as opposed to protein degradation which involved extensive cleavage of the peptide bonds in the substrate. The enzymes required for the peptide bond cleavage are named proteases which are divided into peptidases and proteinases. Peptidases are exopeptidases which hydrolyze single amino acids from the amino-terminus or the carboxy-terminus of a peptide chain. In contrast, the proteinases (also called proteolytic enzymes or endopeptidases) are capable of selectively recognizing and cleaving specific peptide bonds in substrates.
Proteinases are further subdivided into four classes based on the identity of their catalytic amino acid residues, whose relative three-dimensional positions are conserved within a group, and the mechanism of catalysis. The four types of proteinases are: serine, cysteine (thiol), aspartic (acid) and metallo-. Serine proteinases possess a catalytic triad of aspartic acid, histidine and serine residues, and appear to be the most common and widespread type of proteinase. Cysteine proteinases maintain a catalytic diad composed of cysteine and histidine residues in close proximity, whereas the catalytic diad of aspartic proteinases requires two aspartic acid residues. For the metalloproteinases, a divalent cation (usually Zn+2) is required together with essential histidine and glutamic acid residues for catalysis.
Proteinases can be thought of in their most basic form as having a catalytic site, as described above, and a substrate binding pocket. The two sites are usually in close proximity. Generally, proteinases are composed of two globular domains, with amino acids involved in catalysis being contributed by each half of the substrate-binding crevice. For most serine, cysteine and aspartic proteinases, the two globular domains are found within the same polypeptide. However, in the case of the retroviral proteinases, a dimer complex is employed to bring together two individual catalytic centers to form the crevice. Although nearly all substrate-binding crevices achieve a similar three-dimensional structure with respect to the catalytic amino acids for each class of proteinase, the structural conservation does not extend to the substrate binding pocket, which distinguishes a given proteinase from all others. It is this substrate binding region which confers specificity to the proteinase.
It is generally accepted that for the hydrolysis of a specific peptide bond to occur, two requirements must be met. First, the susceptible peptide bonds need to be defined by the nearby amino acid residues with specific side chains which are required for the primary and secondary specificity. The primary specificity has a qualitative feature which targets the selection of the scissile bond, and the secondary specificity conveys a quantitative feature by facilitating the cleavage of the selected bond. Second, the susceptible bond is usually displayed adjacent to the surface of the substrate in a flexible region accessible to the proteinase, and the susceptible peptide must be presented in a three-dimensional conformation which fits the active site pocket of the proteinase. This is referred to as “conformational specificity”.
Many types of post-translational modifications such as phosphorylation, glycosylation and acylation are required for the acquisition and regulation of protein properties such as enzyme activity, protein-protein interactions and intracellular localization. Likewise, limited proteolysis is often used to regulate protein activation or assembly by causing changes in the tertiary structure which bring distant functional amino acid residues together. Interestingly, the free energy required for the reconstruction of the hydrolyzed peptide bond is high and no biological mechanisms for repairing the broken peptide bond have yet been identified. Thus the changes introduced into substrates by proteolytic cleavage are essentially irreversible. This combination of cleavage specificity and reaction irreversibility have resulted in the common utilization of the proteolytic processing reaction as a unidirectional mechanism for a wide variety of biological processes including food digestion, signal peptide cleavage, signal transduction, peptide hormone/growth factor production, blood clotting, complement pathway cascade, pathogen elimination, cell migration and reproduction.
For many plant and animal viruses, a successful infection is dependent on proteolytic processing at one or more stages. In fact, it is the exceptional virus that does not require proteolytic processing during its replication cycle. The required proteolytic enzymes can be provided by either the host cell, the infecting virus, or both. Proteinases provided by the host cell generally contribute to the processing of membrane or envelope proteins that are trafficking through the secretory compartment of the cell. It is within these secretory compartments that viral envelope proteins undergo maturation by cleavage of signal peptides (in addition to acylation and glycosylation), such as the E1 and E2 glycoproteins of Sindbis virus. On the other hand, the proteinases which are responsible for the proteolytic processing of viral proteins are usually encoded by the viruses themselves.
Proteolytic cleavage of viral polypeptides has been categorized as “formative” or “morphogenic” proteolysis, depending on the function the reaction serves during the replicative cycle. Formative proteolysis refers to the processing of viral polyproteins into structural and non-structural protein products. A number of viral formative cleavage proteinases have been identified and are encoded by animal viruses such as picomaviruses, flaviviruses, alphaviruses, retroviruses and coronaviruses. Formative proteolysis provides a mechanism for viruses, such as retroviruses and positive-strand RNA viruses, to utilize a single RNA template for the expression of several viral proteins from a large polyprotein precursor. Morphogenic proteolysis refers to the cleavage of viral structural proteins assembled in previrions during virion maturation. Morphogenic cleavage occurs in conjunction with virion assembly and is often required for the acquisition of infectivity of both DNA and RNA viruses such as picomaviruses, alphaviruses, retroviruses, adenoviruses and bacteriophage T4. Although less is known about morphogenic proteolysis, several different functions have been proposed for this process, including: facilitation of correct genomic RNA dimerization in assembling retroviral particles; unidirectional packaging of bacteriophage T4 DNA; completion of the infectious poliovirus virion in a flexible configuration; and promotion of proper disassembly of adenovirus particles during the initiation of infection.
Regardless of the type of proteolytic maturation reaction employed, it is essential that the activity of the viral proteinases be properly regulated to ensure the efficient production of infectious progeny virions. In general, within biological systems, regulation of proteinases is achieved in several ways, including differential compartmentalization of the enzymes and substrate, presence of specific inhibitors and/or activators, and the proteolytic activation of zymogens. Viruses have adopted similar strategies. For example, in the retroviruses the acidic extracellular environment has been proposed to trigger the morphogenic cleavage of structural proteins by displacing a portion of the gag-pol polyprotein which prevents the active site of the proteinase from interacting with its substrate while within the cell. In the case of adenoviruses, it appears that DNA and a disulfide-linked peptide produced from the pVI structural protein during the latter stages of replication are required for the activation of the viral proteinase and subsequent virus maturation. Finally, perhaps the most elegant example of regulating viral proteinase activity is provided by the core protein of Sindbis virus which undergoes autoproteolysis to become inactive after assembly of the nucleocapsid. Inactivation of the proteinase activity is accomplished by locating the carboxy-terminal region of the protein into the catalytic pocket in concert with the proteolytic cleavage event.
VV Replication Cycle and Postranslational Modification of Viral Gene Products.
Given the importance of limited proteolysis as a means to regulate gene expression in biological systems, and the extent and diversity of ways that even simple viral systems apparently employ this regulatory mechanism, it is of interest to consider if and how a complex virus such as vaccinia virus might incorporate this process into its replicative cycle. Vaccinia virus (VV) is the prototype of the Poxviridae, a family of DNA viruses distinguished by their unique morphology and cytoplasmic site of replication.
The 191 Kbp VV DNA genome encodes at least 263 gene products whose expression is regulated in a temporal fashion during the viral replicative cycle that begins with entry of the virus into the host cell and terminates with the assembly of complex macromolecular structures to form an infectious particle. Unlike many other viruses, VV produces a multiplicity of virion forms, all of which appear to be infectious. Although the molecular details of poxvirus assembly and differentiation remain sketchy and controversial, the most widely accepted scenario of events which transpire is as follows. After (or concurrent with) viral DNA replication, assemblages of progeny DNA molecules, virion enzymes and structural proteins coalesce to form pre-virion particles. These particles acquire two membranes by budding through the intermediate compartment (between the endoplasmic reticulum and the Golgi) to become infectious intracellular mature virus (IMV). A portion of the IMV then becomes enveloped by two additional membranes derived from the trans-Golgi network to form intracellular enveloped virus (IEV). Following migration to the cell surface the outermost IEV membrane fuses with the plasma membrane to give rise to extracellular enveloped virus (EV). The EV can either remain associated with the cell (cell-associated enveloped virus, CEV) or be released into the external medium as extracellular enveloped virus (EEV). Some poxviruses, such as cowpoxvirus (CPV), produce yet another virion form. In CPV-infected cells, large inclusion bodies are produced which are composed primarily of a single 160 kDa viral protein. Within these A-type inclusions are occluded (and infectious) virions.
Considering the large number of viral encoded proteins, the multiplicity of VV virion forms and the number of distinct intracellular sites used during the viral assembly and morphogenesis process, one would predict that VV might utilize a number of the cellular protein modification and targeting pathways to regulate these complex processes. Indeed, it has been demonstrated that during the course of viral replication, VV proteins are matured by a number of posttranslational modifications including acylation, phosphorylation, glycosylation, ADP-ribosylation, and proteolytic processing. Although the details about what role limited proteolytic reactions might play during VV replication were not available when our studies were initiated in 1990, the information that was available in the literature suggested that both formative and morphogenic cleavage pathways might be employed. For example, both the VV growth factor (VGF) and hemagglutinin (HA) proteins appeared to have signal peptides removed via formative proteolysis during their transit through the endoplasmic reticulum and transport to the plasma membrane. Likewise, three of the major structural proteins found within the mature VV virion core, 4a, 4b and 25K, were known to be produced from higher molecular weight precursors at late times during infection, making them candidates for morphogenic cleavages. It was this latter question, namely the nature of the processing reaction by which the major VV core proteins are matured, that the experiments conducted in our laboratory over the last decade have addressed.
VV Proteolysis—What was Known Prior to 1990.
The genes expressed at late times during a VV infection (i.e., those expressed after the initiation of viral DNA synthesis) include most of the structural proteins required for the assembly of progeny virions. The first indication that some of the VV structural proteins might be subject to proteolytic processing occurred when Holowczak and Joklik noted differences in the apparent molecular weights of radioactively-labeled proteins present in VV-infected cells when compared to those found in purified virions. Subsequently, pulse-labeling of VV-infected cells was used to demonstrate that a large precursor protein could be chased into a smaller polypeptide, with concomitant disappearance of the larger-sized protein. This conversion could be specifically inhibited by rifampicin with no apparent effect on the synthesis of the precursor. The precursor protein was designated as P4a and the proteolytically processed product called 4a.
Additional pulse-chase experiments revealed that several other VV structural polypeptides, in addition to P4a, were apparently cleaved during the late phases of the VV replication cycle. These proteolytically processed proteins, referred to using the Sarov and Joklik designation of virion proteins, included 4a, 4b, VP8(referred to as 25K in our work), 9 and 10. This may in fact represent an underestimate of the number of VV late proteins which are subject to proteolysis. The VV core proteins 4a, 4b, and VP8 (25K) are the most abundant proteins in the VV particle, together constituting about 33% of the mass of the virion.
Tryptic peptide mapping and immunological reagents have been used to establish the relationships between the P4a, P4b and P25K precursors and their processed products 4a, 4b and 25K. Location of the three loci encoding these genes have been mapped and the nucleotide sequence of their open reading frames determined. With the completion of the sequence of the entire genome of the Copenhagen strain of VV, the genes encoding the P4a, P4b and P25K precursors received the designations A1OL, A3L and L4R, respectively. The proteolytic processing of VV structural proteins appears to be essential for the formation of infectious progeny virions. This conclusion stems from the observation that there are a variety of different drug treatments (e.g. rifampicin and α-amanitin) or conditional-lethal mutations in the genome, which apparently affect proteolysis (and particle maturation) without affecting overall protein synthesis.